Muscle Structure and Function Recovery: Adalimumab-Calcium Channel Synergy in Post-Ischemic Stroke Sarcopenia

Abstract

Background: Adalimumab, a TNF-α inhibitor, is widely used clinically. Recent studies suggest Adalimumab can improve muscle damage after ischemic stroke (IS), but its protective mechanisms remain unclear. This study investigates the effect of adalimumab on muscle structure post-IS and the role of calcium balance in muscle strength, while validating their synergistic effect.

Methods: This study investigates the effects of adalimumab and GV-58 on muscle structure and function in male middle cerebral artery occlusion (MCAO) rat models using behavioural, imaging, pathological and WB experiments. In vitro mechanisms are explored with L6 and primary muscle cells.

Results: The results of the present study showed a significant decrease in motor function in IS-induced sarcopenia (ISS) rats, as evidenced by shortened length (-47.56%, p < 0.001), reduced weight (-43.79%, p < 0.001) and reduced cross-sectional area of myofibroblasts (-38.58%, p < 0.001) in the soleus muscle as compared to the sham group. Inflammatory factors such as IL-1β, IL-6, TNF-α and reactive oxygen species (ROS) levels were significantly elevated in the muscles of ISS rats (3.10-fold, 3.78-fold, 2.29-fold, 2.80-fold, p < 0.001). Molecular mechanism studies showed that TNF-α, MAFbx and MuRF1 protein expression was down-regulated, and IL-10 and MyoD1 expression was up-regulated in muscle tissues of ISS rats. RNA-seq implicated the Ca²⁺ signalling pathway in ISS-related muscle weakness. Muscle strength in ISS rats is associated with Ca²⁺ content and Ca²⁺ channels, and key excitation-contraction coupling proteins SERCA2, Cav1.1 and RYR1 expression was decreased, whereas Ca²⁺ sensing proteins STIM1 and CAM expression were compensatory upregulated. Adalimumab treatment significantly reduced muscle inflammation and structural damage in ISS rats, significantly increasing the length (+66.88%, p < 0.001) and weight (+43.92%, p < 0.001) of the soleus muscle and increasing muscle cell cross-sectional area (+53.44%, p < 0.001). Adalimumab also inhibited the expression of MAFbx, MuRF1 and promoted the expression of IL-10 and MyoD1. GV-58 treatment of L6 cells showed that combined administration with adalimumab produced a synergistic effect. Upregulation of key Ca²⁺ protein expression such as RyR1 and SERCA1 improved the recovery of muscle strength in ISS rats while maintaining muscle structure.

Conclusions: The combination of adalimumab and GV-58 effectively restores muscle function after stroke by inhibiting inflammation and improving calcium channel dysfunction.

Keywords: Ca2+ channels; adalimumab; combination therapy; ischemic stroke; muscle structure.

FIGURE 1

Characterization of ischemic injury and skeletal muscle dysfunction in MCAO rats. (a) Representative images of laser speckle in rat brains and analysis of blood flow perfusion on days 1, n = 6. (b) Triphenyltetrazolium chloride staining of brain sections on day 7, n = 5. (c) Zea‐Longa neurological score and bederson score assessment of motor impairment. (d) Analysis of body weight, forelimb grip strength, and movement distance in rats. (e) Images of soleus muscle from each group and quantification of soleus muscle length and weight, n = 5. (f) H&E staining of soleus muscle showing morphological alterations, scale bar = 50 μm, n = 5. *p < 0.05, **p < 0.01, ***p < 0.001.

FIGURE 2

Adalimumab mitigates inflammation and muscle loss in MCAO rats. (a–c) Quantification of proinflammatory cytokines IL‐6, IL‐1β, and TNF‐α in the soleus muscle, n = 5. (d) Flow cytometry analysis of reactive oxygen species (ROS) levels in muscle tissue after MCAO, n = 4. (e) Representative images of muscle electrical signals in rats and intensity analysis, n = 5. (f) Representative ultrasound imaging of muscle morphology and thickness analysis, n = 5. *p < 0.05, **p < 0.01, ***p < 0.001.

FIGURE 3

Effects of adalimumab on muscle electrophysiology, ultrastructure, and molecular regulation in MCAO rats. (a) IHC staining and quantification of TNF‐α, MuRF1 and MAFbx expression in the soleus muscle, scale bar = 50 μm. (b) Transmission electron microscopy images of the soleus muscle and corresponding ultrastructural scale bar = 500 nm. (c) WB and relative expression analysis of TNF‐α, IL‐10, MyoD1, MuRF1 and MAFbx in the soleus muscle, n = 4. (d) Co‐IP analysis confirming molecular interactions among TNF‐α, MuRF1 and MAFbx. (e‐f) CETSA evaluating the effect of adalimumab on the thermal stability of TNF‐α, MuRF1 and MAFbx. *p < 0.05, **p < 0.01, ***p < 0.001.

FIGURE 4

Transcriptomic analysis and calcium signalling‐related molecular changes in the soleus muscle of MCAO rats. (a) Principal component analysis demonstrating sample clustering and variation between experimental groups. (b) KEGG pathway enrichment analysis highlighting key signalling pathways associated with DEGs. (c) Gene ontology analysis illustrating major altered biological processes. (d) Volcano plot depicting the distribution of upregulated and downregulated DEGs. (e) FC analysis of intracellular Ca²⁺ levels in soleus muscle tissue of MCAO rats, n = 4. (f) Representative images of Alizarin Red staining of the soleus muscle, scale bar = 50 μm. *p < 0.05, **p < 0.01, ***p < 0.001. DEGs, differentially expressed genes.

FIGURE 5

Altered Ca²⁺ channel modulatory proteins lead to poststroke muscle impairment. (a–b) IHC staining and quantification of Cav1.1 and RyR1 expression in the soleus muscle, scale bar = 50 μm, n = 4. (c) WB bands and quantitative analysis of Ca²⁺‐regulatory and muscle atrophy‐related proteins, including CaM, STIM1, Cav1.1, SERCA2, Troponin I, MyoD1, MuRF1 and MAFbx, n = 3. (d) Representative IF images showing MyoD1 and phalloidin expression at different time points following adalimumab treatment, scale bar = 28.1 μm. (e) WB bands and corresponding quantitative analysis of TNF‐α, IL‐10, MyoD1, MuRF1, MAFbx, CaM, STIM1, CASQ1, RyR1 and calpain 1 in primary muscle cells, n = 4. *p < 0.05, **p < 0.01, ***p < 0.001.

FIGURE 6

Synergistic treatment with adalimumab and Gv‐58 activates Ca²⁺ regulatory pathways and suppresses muscle atrophy markers. (a) Cell viability analysis following GV‐58 treatment. (b) Effects of GV‐58 on TNF‐α expression levels in primary cells. (c–d) Time‐dependent IF images showing changes in cytosolic and mitochondrial Ca²⁺ levels after GV‐58 and adalimumab co‐treatment, scale bar = 17.9 μm, scale bar = 22.7 μm. (e) Representative WB bands and expression analysis of RyR1, SERCA2, TNNC2, CASQ1, Annexin A2, calpain 1, MyoD1, MuRF1, MAFbx, Myostatin and MYH1 in response to combination therapy at different time points, n = 6. *p < 0.05, **p < 0.01, ***p < 0.001.

FIGURE 7

Effects of combined treatment with GV‐58 and adalimumab on muscle function and structure in MCAO rats. (a) Zea‐Longa neurological scores following combination therapy. (b–c) Quantitative analysis of locomotor distance and forelimb grip strength. (d) Representative images and quantification of soleus muscle length and weight after intervention, n = 5. (e) Representative H&E‐stained sections showing histological changes in soleus muscle, scale bar = 50 μm. (f) Representative images of muscle electrical signals in rats and intensity analysis, n = 5. *p < 0.05, **p < 0.01, ***p < 0.001.

FIGURE 8

Effects of combined treatment with GV‐58 and adalimumab on muscle Ca²⁺ homeostasis in MCAO rats. (a) IHC staining and quantification of RyR1 and SERCA1 expression in soleus muscle, scale bar = 50 μm, n = 4. (b) FC analysis of intracellular Ca²⁺ levels in skeletal muscle. (c–d) WB detection of Annexin A2, TNN2, Calsequestrin 1, STIM1, MyoD1, MuRF1 and MAFbx expression following combination treatment, n = 4. *p < 0.05, **p < 0.01, ***p < 0.001.